The World Health Organization (WHO) estimates that 8.9 million cases of TB occurred in 2004, with 3.9 million of these representing the most infectious, smear-positive, form. Of the estimated 1.7 million deaths, over 260,000 occurred in HIV-infected individuals; the increasing incidence of TB worldwide is due almost entirely to cases in areas of Africa with a high prevalence of HIV. Over 424,000 of the 2004 cases were multidrug resistant (MDR), i.e., resistant to isoniazid (INH) and rifampin (RIF); most occurred in China, India, and the Russian Federation. Among MDR cases, approximately 10% are extensively drug resistant (XDR), i.e., also resistant to three or more of the six classes of second-line drugs. MDR- and XDR-TB pose the greatest threats to public health in the resource-poor areas mentioned above, and WHO has recently emphasized the need for inexpensive, rapid, and simple TB diagnostic and antimicrobial susceptibility tests suitable for use in such areas. The microscopic observation direct susceptibility (MODS) assay has attracted considerable attention in this context. Decontaminated sputum is inoculated into multiwell plates containing liquid medium, with first-line drugs (typically INH and RIF) in some wells, and the wells are observed over a period of days. Growth of Mycobacterium tuberculosis (MTB) is detected by the cordlike appearance of colonies under a low-power microscope. The assay, uses inexpensive consumables and simple equipment, and determines culture positivity and antibiotic susceptibility at a median of 7 days; modern automated methods, employing expensive equipment and consumables, and classical culture techniques are less sensitive and take longer. This project will investigate the use of a small, simple, extremely inexpensive fluorescence image cytometer to automate the readout of the MODS assay or develop a functional equivalent, in either case eliminating the need for a trained microscopist, with the anticipated result being an accurate, rapid, and sensitive apparatus and method for detecting TB and determining antibiotic susceptibility, suitable for use in developed as well as developing countries. PUBLIC HEALTH RELEVANCE: An estimated 8.9 million cases of tuberculosis (TB) occurred in 2004; of the estimated 1.7 million deaths, over 260,000 occurred in HIV-infected individuals. Multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) TB are a significant threat to public health in resource- poor areas such as Africa, China, India, and the Russian Federation, where epidemics of TB and HIV-AIDS interact in a negative fashion. The World Health Organization has recently emphasized the need for inexpensive, rapid, and simple TB diagnostic and drug susceptibility tests suitable for use in such areas. One such test, the MODS assay, is rapid and inexpensive, but requires readout by a skilled microscopist. This project will attempt to develop an inexpensive apparatus, using simple optics, light-emitting diodes, and a digital camera chip, that could be used for an automated MODS assay or its functional equivalent, meeting diagnostic needs in both resource-poor and affluent countries. [unreadable] [unreadable] [unreadable]